Li Y X, Papkoff J, Sarkar N H
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Virology. 1999 Mar 1;255(1):138-49. doi: 10.1006/viro.1998.9571.
Activation of the protooncogene Wnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice. Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state. Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuous Wnt-1 expression for their ability to form tumors in vivo. To address this question, we constructed antisense and sense Wnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenous Wnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and sense Wnt-1 RNAs, changes in endogenous Wnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cells in vitro and in vivo were also examined. Our results show that (1) the levels of the expression of endogenous Wnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisense Wnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors ( approximately 0.2 cm in 16 weeks) compared to the tumors ( approximately 2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest that Wnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity.
已知通过插入小鼠乳腺肿瘤病毒（MMTV）激活原癌基因Wnt-1可在小鼠体内引发乳腺肿瘤。据推测，乳腺中Wnt-1的表达可直接刺激乳腺上皮细胞局部生长，使其进入肿瘤前状态。Wnt-1的表达还可诱导培养的正常乳腺细胞发生形态改变。然而，转化的乳腺细胞在体内形成肿瘤的能力是否需要持续的Wnt-1表达尚未确定。为解决这个问题，我们构建了反义Wnt-1表达载体和正义Wnt-1表达载体，它们含有一个由五个高亲和力糖皮质激素反应元件（GRE5）组成的合成启动子。该启动子对地塞米松的诱导能力比MMTV长末端重复序列中所含的启动子至少高50倍。将这些载体导入一个小鼠乳腺肿瘤细胞系（R/Sa-MT），该细胞系表达高水平的内源性Wnt-1 mRNA，移植到同基因宿主中时会形成快速生长的肿瘤。在建立的12个稳定转染细胞系中（9个为反义构建体，3个为正义构建体），检测了2个反义细胞系（R/Sa-MT/反义）和1个正义细胞系（R/Sa-MT/正义）对地塞米松诱导反义Wnt-1 RNA和正义Wnt-1 RNA的能力、内源性Wnt-1 RNA表达的变化以及细胞形态的变化。还检测了这些细胞在体外和体内的生长模式。我们的结果表明：（1）与R/Sa-MT/正义细胞和R/Sa-MT对照细胞相比，在暴露于地塞米松后高水平表达反义Wnt-1 RNA的那些细胞（R/Sa-MT/反义）中，内源性Wnt-1 mRNA和蛋白质的表达水平显著降低（>80%）；（2）与R/Sa-MT/正义细胞和R/Sa-MT细胞产生的肿瘤（8周时约2.0 cm）相比，R/Sa-MT/反义细胞移植产生的肿瘤较小（16周时约0.2 cm）。因此，我们认为Wnt-1的表达不仅是正常乳腺细胞转化为肿瘤细胞所必需的，也是维持其致瘤性所必需的。
